1. Field of the Invention
This invention relates to surface modification of ferromagnetic chromium dioxide particles. The modified particles are useful in heterogeneous diagnostic assays and bioaffinity separations.
2. Background Art
The concept of using magnetically responsive particles to effect separations of bioactive materials is old in the art (Hedin, C. G., Biotech. Bioeng. Symp. No. 3 (1972) 173-174; Robinson, P. J., et al, Biotech. Bioeng. (1973) 15, 603-606). The concept has been extended over time to include affinity purification of enzymes, proteins or microorganisms applicable to any sorption-desorption process (Dunhill, P., et al, Biotech. Bioeng. (1974) 10, 987-990; Horisberger, M., Biotech. Bioeng. (1976) 18, 1647-1651).
Hersh, L. H., et al describe in U.S. Pat. No. 3,933,997 the first use of magnetically responsive particles as the solid support in heterogeneous immunoassays. The preferred particle described is a ferrous oxide particle; however, CoO, NiO, Mn.sub.2 O.sub.3, CoMnP particles are also disclosed and claimed.
Subsequently, many varieties of magnetically responsive particles have been described. Ithakissios in U.S. Pat. No. 4,115,534 discloses the use of composite microparticles employing a permeable, solid, water-insoluble matrix comprising a proteinaceous material, a polysaccharide, a polyurethane or mixtures thereof. A variety of ferromagnetic substancess may be employed, BaFe.sub.12 O.sub.19, Fe.sub.3 O.sub.4, CoO, NiO, Mn.sub.2 O.sub.3, CoMnP, iron or nickel, according to this invention.
Forrest et al in U.S. Pat. No. 4,141,687 describe and claim the use of composite microparticles which have a specific gravity near that of the reaction millieu. This improvement allows the particles to remain suspended in the solution allowing for more efficient capture kinetics. Again, a variety of ferromagnetic substances are described and claimed as part of this invention. This list includes CrO.sub.2 along with iron, magnetic iron oxides, nickel or cobalt. However, the density of CrO.sub.2 particles, 4.85, would limit the number of particles which could be incorporated into the composite matrix. Microparticles thus prepared would be magnetically very dilute, requiring high gradient field strengths for separation.
A still further improved magnetically responsive particle is described by Mansfield et al in U.S. Pat. No. 4,197,337. These particles are porous glass microparticles with magnetic material imbedded within them. This gives the particles the properties of high surface area, inertness and being substantially superparamagnetic. This high surface area again favors rapid reaction kinetics and increases capacity of the individual particles. Being substantially superparamagnetic basically means the particles do not retain much magnetic memory, or rentitivity, when removed from a magnetic field. This means that particles can be repeatedly separated in a magnetic field without affecting the ability to redisperse those particles. This is of advantage in sandwich immunoassays where multiple washing steps may require repeated separation and redispersion. These particles are of necessity magnetically dilute and thus also require relatively high field strengths for separation.
Perhaps the most recent improved magnetic particle is that described by Chagnon et al in Danish Application DK No. 2374/84 and commercially available under the trade name Biomag.RTM. from Advanced Magnetics. These particles are composed of a cluster of microcrystals bound together by a silane coating. These particles have a diameter in the range 0.1 to 1.5 .mu.M with a very high surface area, 100-150 m.sup.2 /g, a settling time of greater than 1.5 hours and a magnetic separation time of less than 10 minutes. The long settling time of the particles favors rapid reaction kinetics. The microcrystal size of Biomag.RTM. is also substantially smaller than those described by Mansfield; thus, the particles are truly superparamagnetic which eliminates the problem of magnetic aggregation. The metal oxide is defined as a group of crystals consisting of transition metal oxide with "ferro-spinel" structure which excludes rutile structure CrO.sub.2. Indeed, the teaching of DK No. 2374/84 would not be applicable to CrO.sub.2 particles because it would not adequately protect the CrO.sub.2 from hydrolysis. This invention provides particles which take advantage of the favorable magnetic properties of CrO.sub.2 in a composition in which the CrO.sub.2 is protected from hydrolysis.
The protected CrO.sub.2 particles of this invention have the following properties:
low remanent magnetism and favorable surface structure--allowing repeated magnetic separation/dispersion cycles;
rapid separation in a magnetic field;
high surface area for high capture capacity;
a highly stable particle for maximum reagent shelf life.
It is known that CrO.sub.2 is ferromagnetic and crystals or particles of CrO.sub.2 are useful in the production of magnetic recording tapes. However, magnetic recording applications have requirements quite different from immunoassays, specifically, in a hydrolytic particle stability and particle size.
It is well known that the hydrolytic stability of CrO.sub.2 crystals is poor even relative to the needs of magnetic tape where the crystals are bonded to a polymer (relatively) isolated from water. In U.S. Pat. No. 3,512,930, Bottjer and Ingersoll teach a reductive surface treatment of CrO.sub.2 to improve its stability. In this process the surface of the CrO.sub.2 crystal is reduced to form a protective layer which is much more hydrolytically stable. This coating, however, is easily reoxidized by air. The requirements for resistance to air oxidation are much greater for immunoassay applications than for tape applications so the particles while suitable for tape applications are not suitable for use as solid supports in immunoassays. Also of note is that the reduced surface of the crystal is nonmagnetic, thus a minimal coat is used in tape applications. As will be shown below a thick coating reducing the magnetic properties of the particle is advantageous in immunoassay applications.
Further means of stabilization of CrO.sub.2 has been described such as that in U.S. Pat. No. 4,275,114 issued to Schoenafinger. This patent teaches the use of a block copolymer of siloxane units and alkyleneoxy units. Schoenafinger does not consider stability in aqueous solution as would be necessary for immunoassay use nor does he suggest that the particles described would be useful for such applications.
Another alternate approach to surface stabilization of CrO.sub.2 particles is disclosed in U.S. Pat. No. 4,068,038 in which one or more insoluble or sparingly soluble cationic or anionic compounds such as Mg, Zn, Ce, La, Fe, Mn and Co with MoO.sub.4 (-2), WO.sub.4 (-2) or PO.sub.4 (-3), or hydrated or unhydrated oxides or hydroxides of amphoteric metals such as silicon, titanium or tin deposited on the surface. This means of stabilization has the advantage of retaining more of the high ferromagnetic properties desired for tape applications.
It is known that a dense silica coating improves the opacity and dispersibility of TiO.sub.2 particles for use as a pigment, U.S. Pat. No. 3,427,507. It is also known that CrO.sub.2 is isostructural with TiO.sub.2. As described below encapsulating the surface reduced CrO.sub.2 in silica provides significant advantages in immunoassay applications.
In order for any magnetic particle to be useful as a solid support in heterogeneous immunoassays, it must be derivatised. Since the original teachings of Hersh et al the use of functionalized silanes has been a preferred intermediate between the particle and the bioactive protein. Processes such as those described by Weetall, H. H. in U.S. Pat. No. 3,652,761 are illustrative of this procedure. The functionalized silanes can then be linked to bioactive reagents either directly, with homobifunctional or heterobifunctional crosslinkers.
Another useful characteristic for a magnetic particle to be used in immunoassay is a relatively high, but well controlled, particle size distribution. U.S. Pat. No. 4,524,008 issued June 18, 1985, to Chen describes an improved method for controlling CrO.sub.2 particle size.
We have found that CrO.sub.2 particles exceptionally well suited to use in immunoassays and other applications constituting bioaffinity separations can be produced using a multilayer coating process to stabilize and functionalize particles.